Myles H. Akabas

Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins

The nicotinic acetylcholine (ACh) receptors and the other neurotransmitter-gated ion channels have key roles in fast synaptic transmission throughout the nervous system. These receptors have a simple functional repertoire: they bind a specific neurotransmitter, open a gate, conduct specific ions across the membrane, and desensitize. Although it is easy for us to imagine in a general way how they might carry out these steps, to determine the actual mechanism requires more detailed structural information than we now have. Nevertheless, new information about the parts of these receptors that are in the front line of function, the neurotransmitter binding sites, the ion-conducting channel , and the gate, provides intriguing clues, albeit not always consistent ones, about the mechanisms of these receptors. Most progress toward understanding function in terms of structure has been made with the ACh receptors , on which we will focus, at the same time noting what is conserved and what is variable among all of the neuro-transmitter-gated ion channels. The ACh receptors are members of a family of neuro-transmitter-gated ion channels, which also includes receptors for y-aminobutyric acid (GABA), glycine (Gly), and 5hydroxytryptamine in this family is an invertebrate glutamate-gated chloride channel (Gully et al., 1994). The subunits of these receptors have similar sequences and distributions of hydropho-bic, membrane-spanning segments and are homologous (Figures 1 and 2). In this family, each subunit contains, in its N-terminal extracellular half, 2 cysteine (Cys) residues separated by 13 other residues. These Cys residues are disulfide linked in the ACh receptor (Kao and Karlin, 1986) and presumably in the homologous receptors, thereby closing a 15-residue loop. Because of this unique, invariant feature, we will call this family the Cys-loop receptors. The subunits of other ligand-gated ion channels-the receptors for glutamate (cation-conducting), for ATP, and for the second messengers, CAMP and cGMP-have sequences and distributions of putative membrane-spanning segments that are dissimilar from those of the Cys-loop receptors (Figures 1 and 2). Despite the differences in their structures, all of these ligand-gated ion channels carry out the same general functions. This implies that, at the level of detailed mechanisms, there are many ways of recognizing specific ligands, of transducing binding into propagated structural changes, of gating a channel, and of selecting and conducting specific ions through a membrane. It is likely, however, that among the homologous Cys-loop receptors the mechanisms are very similar and that insight into one is applicable to all. Overall …

SUBSTITUTED-CYSTEINE ACCESSIBILITY METHOD

Publisher Summary The functional properties of ion channels, such as gating, ion selectivity, single-channel conductance, multi-ion occupancy, transient blocking, desensitization, and inactivation, have been extensively studied by eleclrophysiologic techniques. The molecular bases for these functions are being gradually revealed by protein chemical methods, by genetic manipulation, and by direct structural determinations. This chapter describes an approach to channels that combines chemical and genetic approaches. The substituted-cysteine accessibility method (SCAM) can be used to identify all of the residues that line a channel, to size the channel, to determine differences in the structures of the channel in different functional states, to locate the gates and selectivity filters, and to map the electrostatic potential profile in the channel. It describes the synthesis and properties of some thiosulfonate derivatives, and it also describes applications of SCAM to ion channels, to transport proteins, and to the membrane-embedded binding sites of G-protein-coupled receptors.

Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit

Each residue in and flanking the M2 membrane-spanning segment of the alpha subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Cysteines substituted for Glu-262, Leu-258, Val-255, Ser-252, Leu-251, Leu-250, Ser-248, Leu-245, Thr-244, and Glu-241 reacted with the positively charged, hydrophilic, sulfhydryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), added extracellularly. These 10 residues, therefore, are exposed in the channel lumen. The pattern of exposure is compatible with an alpha helix, interrupted by an extended structure from Leu-250 to Ser-252. Acetylcholine caused subtle changes in the accessibilities of some of the engineered cysteines. Since all 10 residues are accessible to MTSEA in the closed state of the channel, the channel gate is at least as cytoplasmic as Glu-241, the most cytoplasmic of the residues tested.

Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit.

The muscle-type acetylcholine (ACh) receptor has the composition alpha 2 beta gamma delta. The subunits are arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the alpha M2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substituted-cysteine-accessibility method to alpha M1. We analyzed 15 consecutive residues, starting at alpha Pro211 at the extracellular end of M1. Wild-type alpha contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant alpha subunits, together with wild-type beta, gamma, and delta subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: alpha Y213C was susceptible to MSTEA added in the presence or the absence of ACh, alpha P211C, alpha I215C, alpha V216C, alpha N217C, and alpha I220C were susceptible in the absence of ACh, and alpha V218C was susceptible in the presence of ACh. These results imply that M1 is exposed in the channel, and its exposure changes during gating or desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants

We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257-261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 1 subunit. The residues alpha 1Val257 and alpha 1Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of alpha 1V257C and alpha 1T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as alpha 1Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to alpha 1T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Co-application of picrotoxin prevented the sulfhydryl reagents from reacting with alpha 1V257C but did not prevent reaction with the more extracellular residue alpha 1T261C. Picrotoxin protection of alpha 1V257C may be due to steric block by picrotoxin bound in the channel at the level of alpha 1Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which alpha 1V257C was accessible to sulfhydryl reagent.

Exposure of Residues in the Cyclic Nucleotide–Gated Channel Pore: P Region Structure and Function in Gating

In voltage-gated ion channels and in the homologous cyclic nucleotide-gated (CNG) channels, the loop between the S5 and S6 transmembrane segments (P region) is thought to form the lining of the pore. To investigate the structure and the role in gating of the P region of the bovine retinal CNG channel, we determined the accessibility of 11 cysteine-substituted P region residues to small, charged sulfhydryl reagents applied to the inside and outside of membrane patches in the open and closed states of the channel. The results suggest that the P region forms a loop that extends toward the central axis of the channel, analogous to the L3 loop of bacterial porin channels. Furthermore, the P region, in addition to forming the ion selectivity filter, functions as the channel gate, the structure of which changes when the channel opens.

Cystic Fibrosis Transmembrane Conductance Regulator STRUCTURE AND FUNCTION OF AN EPITHELIAL CHLORIDE CHANNEL

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a Cl channel that is an essential component of epithelial Cl transport systems in many organs, including the intestines, pancreas, lungs, sweat glands, and kidneys. In the Cl secretory intestinal epithelium, Cl enters the cells through a Na-K-2Cl cotransporter in the basolateral membrane and exits through CFTR in the apical membrane; water follows osmotically (1). Absorptive epithelia use similar transporters and channels, but their polarized distribution between the apical and basolateral membranes is usually reversed. A major determinant of the transepithelial Cl transport rate is the level of activation of CFTR (2, 3), which depends on the extent to which it is phosphorylated. This is determined by the relative activities of kinases and phosphatases, the activities of which are often hormonally regulated (1). Defects in the gene encoding CFTR that reduce either its Cl transport capacity or its level of cell surface expression cause cystic fibrosis (CF) (4–6) as well as a form of male sterility due to congenital bilateral absence of the vas deferens (7). CF is the most common lethal genetic disease in Caucasians, with about 30,000 CF patients in the United States. In contrast, in intestinal epithelial cells overstimulation of CFTR because of the activation of protein kinases by bacterial enterotoxins causes secretory diarrhea (1, 8). Secretory diarrhea is the second largest cause of infant mortality in the developing world, causing 3 million deaths per year of children under the age of 5. Thus, although CFTR was named because of its association with CF, as a cause of disease, its relationship to secretory diarrhea is a more widespread public health problem. The cloning of CFTR in 1989 (9) has facilitated studies of its structure, function, regulation, biogenesis, and degradation, which will be reviewed in this article. Issues reviewed elsewhere and not discussed here include the mechanisms by which mutations in CFTR cause CF (5, 6) and the possible role of CFTR in regulating the pH within intracellular organelles (10).

Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.

γ-Aminobutyric acid increases the water accessibility of M3 membrane- spanning segment residues in γ-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.

PROBING THE STRUCTURAL AND FUNCTIONAL DOMAINS OF THE CFTR CHLORIDE CHANNEL

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channels functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely α-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.